EXCLI J EXCLI Journal 1611-2156 Leibniz Research Centre for Working Environment and Human Factors 2022-5141 10.17179/excli2022-5141 Doc1171 Original article Association between expression of ZBP1, AIM2, and MDA5 genes and severity of COVID-19 Arefinia Nasir 1 Ramezani Amin 2 3 Farokhnia Mehrdad 4 Arab Zadeh Ali Mohammad 4 Yaghobi Ramin * 5 Sarvari Jamal 1 6 Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran Department of Internal Medicine, School of Medicine, Research Center for Hydatid Disease in Iran, Afzalipour Hospital, Kerman University of Medical Sciences, Kerman, Iran Transplant Research Center, Department of Pathology, Shiraz University of Medical Sciences, Shiraz, Iran Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran *To whom correspondence should be addressed: Ramin Yaghobi, Transplant Research Center, Department of Pathology, Shiraz University of Medical Sciences, Shiraz, Iran; Phone: +9871-42451791, Fax: +98 711 2304069, E-mail: rayaviro@yahoo.com 01 09 2022 2022 21 1171 1183 17 06 2022 22 08 2022 Copyright © 2022 Arefinia et al. 2022

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Antiviral and inflammatory responses following the detection of the virus genome by nucleic acid sensors play a vital role in the pathogenesis and outcome of diseases. In this study, we investigated the ZBP1, AIM2, and MDA5 expression levels in COVID-19 patients with different intensities of the disease. 75 quantitative Real-Time PCR (qRT-PCR)-confirmed COVID-19 patients were included consecutively and divided into 3 groups of mild, severe, and critical based on the severity of the disease. Also, 25 healthy volunteer subjects were included. PBMCs were collected from the whole blood, and RNA was extracted using commercial kit. The expression of ZBP1, AIM2, and MDA5 genes was investigated using qRT-PCR technique. The mean age of the patients and healthy volunteers was 52.73±13.78 and 49.120±12.490, respectively. In each group, 13 out of 25 participants were male. The expression levels of ZBP1 (P=0.001), AIM2 (P=0.001), and MDA5 (P= 0.003) transcript were significantly higher in COVID-19 patients than the control group. The results also revealed that the expression levels of ZBP1, AIM2, and MDA5 were significantly higher in the critical and severe COVID-19 patients compared to those with mild disease (P<0.05). Moreover, regarding the gender, the expression levels of AIM2 and MDA5 were significantly elevated in male severe (P=0.04 and P=0.003, respectively) and critical (P=0.005 and P=0.0004, respectively) patients than the female ones. The results indicated that ZBP1, AIM2, and MDA5 genes might have an important role in the severity of COVID-19 disease. Moreover, the severity of COVID-19 disease in male and female patients might be related to AIM2, and MDA5 expression levels. More studies are recommended to be conducted to clarify this issue.

ZBP1 AIM2 MDA5 SARS-CoV-2 COVID-19 severity of COVID-19
Introduction

The SARS-CoV-2 virus has caused a global pandemic in 2020, infecting millions of people and leading to a global health crisis. COVID-19, a disease caused by SARS-CoV-2, causes not only severe pneumonia and acute respiratory distress syndrome (ARDS) in vulnerable people, but also damage to many organs, including the heart, kidney, pancreas, etc. (Sharif-Zak et al., 2022[37]). It has devastating health effects with high mortality and morbidity worldwide (Guan et al., 2020[11]). The disease is still a mystery and many questions about its pathogenesis remain unanswered. As it is clear, the immune system has an important role in its pathogenicity. Host defense in mammals fights the pathogens through two types of immunity: innate and adaptive immunity (Chaplin, 2010[5]). Intrinsic immunity acts as a pathogenic sensor and helps to eradicate the pathogens and build compatible immunity. These functions are highly dependent on pattern recognition receptors (PRRs) (Ansariniya et al., 2021[2]). PRRs are expressed in many cell types throughout the body and are, therefore, the first sensors likely to detect many viral infections, including SARS-CoV-2 (Kang et al., 2002[18], 2004[17]).

One of the most important interferon-induced PRRs is Z-DNA-binding protein 1 (ZBP1), which functions as a cytosolic nucleic acid sensor and an antiviral immune response modulator (Daniels et al., 2019[7]; Ghoreshi et al., 2022[10]). After activation, ZBP1 recruits the receptor interacting protein kinase (RIPK3) 1 and 3 to execute programmed cell death, including apoptosis, necroptosis, pyroptosis or a mixture of them, depending on the cell type and caspase activity (Thapa et al., 2016[39]). ZBP1 promotes inflammatory signaling and contributes to SARS-CoV-2-induced cytokine production (Bader et al., 2022[3]). It has been reported that ZBP1 proteins are able to induce cell death in response to influenza A virus (IAV) infection (Zhang et al., 2020[50]).

One of cytoplasmic immune system sensors which plays a key role in inflammation is Absent In Melanoma 2 (AIM2) (Junqueira et al., 2021[15]). AIM2 has antiviral responses by sensing the genome DNA of a host mitochondria, the membrane of which was damaged during pyroptosis (Wang et al., 2020[42]). SARS-CoV-2-infected cells undergo programmed cell death mediated by AIM2 inflammasome (Junqueira et al., 2022[14]). In addition, it induces cytokine storm through production of remarkably high level of inflammatory cytokine and eventually reduces the immune response (Liu et al., 2021[23]).

Another cytoplasmic immune system sensor is MDA5. Although first identified in the context of cancer (Kang et al., 2004[17]), it has been shown to have several roles in the host defense against a wide variety of viruses such as coronavirus (Roth-Cross et al., 2008[35]). MDA5 could detect different conformations of RNA, and it is critical for inducing type I interferon (IFN-I) (Zalinger et al., 2015[47]). It has been reported that cytosolic MDA5 is crucial to the induction of IFN type I and III in lung cancer cells following SARS-CoV-2 infection (Sampaio et al., 2021[36]). Moreover, the mice deficient in MDA5 expression demonstrated greater severity of disease when infected with MHV than did wild-type mice (Zalinger et al., 2015[47]).

Therefore, in this study we aimed to compare the expression levels of ZBP1, AIM2, and MDA5 in different stages of COVID-19 disease as well as the healthy control group.

Materials and MethodsSubjects

From February to March 2021, 75 COVID-19 patients were recruited consecutively from Afzalipour Hospital affiliated to the Kerman University of Medical Science. The selected patients were divided into three groups including critical (n=25), severe (n=25), and mild (n=25) according to the World Health Organization (WHO) criteria (WHO, 2021[45]). Inclusion criteria were as follows: 1) patients that met the diagnostic standard of SARS CoV-2 virus based on Centers for Disease Control and Prevention (CDC) definition, as an acute respiratory disease with laboratory-confirmed SARS CoV-2 infection by qRT-PCR (WHO, 2021[45]); 2) Positive results from throat swabs; 3) Patients with intense cough with bloody sputum, purulent sputum, chest pain, severe vomiting, diarrhea, dehydration and dyspnea; 4) Patients with complete clinical information; 5) Patients who did not complain about other infectious diseases, history of hematological diseases or immune system disorders; and 6) Patients who were inoculated with any of the Corona vaccines. In addition, patients under the age of 26 or over 76, pregnant women, and patients with the underlying disorder were excluded from the study. Moreover, 25 age- and gender-matched volunteers, as a healthy control group, were also included. Exclusion criteria for the healthy control group included any underlying chronic viral diseases such as HIV, HBV and HCV, cancers, autoimmune diseases as well as recent bacterial and/or viral infections. A structured questionnaire was used to collect demographic characteristics, temperature, and number of breaths per minute, weekly exercise, smoking, and history of underlying disease. Written informed consent was obtained from each participant, and the study was approved by the local Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.REC.1400.737).

Criteria for the severity of COVID-19

Clinically, patients with the following symptoms were considered critical: 1) continuous high fever for 3 days or more; 2) intense cough with bloody sputum, purulent sputum, or chest CT; 3) rapid respiratory rate, dyspnea and cyanosis in the lips; 4) mind changes including unresponsiveness, somnolence, restlessness, and convulsion; 5) intense vomiting, diarrhea and dehydration; 6) signs of pneumonia examined by radiography; and 7) rapidly increased levels of Lactate dehydrogenase (LDH) and D-Dimer. In our study, patients who met the diagnostic criteria for SARS CoV-2 and also met three of the above conditions were assigned to the severe COVID-19 group (25 cases), and those who met the diagnostic criteria for SARS CoV-2 but not the above conditions were included in the mild COVID-19 group (25 cases).

Blood sampling and PBMC isolation

Totally, 6 mL of blood was collected from each participant in heparin tube. Peripheral Blood Mononuclear Cells (PBMCs) were immediately prepared from the six mL blood diluted with phosphate‐buffered saline (PBS), using Ficoll (Bahar-Afshan, Iran) by the standard density gradient centrifugation, followed by two washes with PBS. Isolated PBMCs pellets were re-suspended in 200 μL of PBS. The cells were counted by a hemocytometer (Neubauer chamber). Aliquots of approximately 2.1 million cells were stored at -80 °C until further analysis.

RNA extraction and real time-PCR

RNA was extracted from PBMCs using the commercial Kit (Favorgen, Taiwan), according to the manufacture instructions. The absorbance of the extracted RNAwas measured at 280 and 260 nm wavelength, using a Nanodrop ND 1000 (Thermo Scientific USA) to determine their quantity and purity. The extracted RNA samples were converted to complementary DNA (cDNA), using cDNA synthesis kits (Yekta-Tajhiz, Iran) according to the manufacturer's instructions. qRT-PCR system (QIAGEN Systems, Germany) was utilized to assess the ZBP1, AIM2, and MDA5 gene expression using SYBR green master mix (Amplicon, United Kingdom). qRT-PCR amplifications with 96 to 106 % efficiencies were set up in triplicate with a 10 μL volume containing 5 μL SYBR green master mix, 1 μL of the corresponding cDNA, and 10 pMol primers (Table 1(Tab. 1)). Amplification efficiencies were calculated and included in data normalization. The qRT-PCR was performed at the following conditions: 94 °C for 15 min; 40 sequential cycles at 95 °C for 10 s, 59 °C, 58.5 °C, 60.4 °C (ZBP1, AIM2 and MDA5, respectively) for 30 sec; and at 72 °C for 25 sec. At 72 °C for five min, the final extension was completed. Analysis of the melt curve confirmed the specificity of qRT-PCR. The β-actin gene was also used as an internal control and the levels of the ZBP1, AIM2, and MDA5 expression were calculated by Pfaffl formula.

Statistical analysis

The SPSS 26 and graphPad Prism 9 software packages were used for data analysis and drawing the graphs. Data were analyzed using independent-samples t-test, nonparametric test following Dunnett's test and two-way ANOVA. P values less than 0.05 were considered as significant.

ResultsDemographic characteristics of the subjects

In this cross-sectional study, 100 subjects which included 75 COVID-19 patients (25 mild, 25 severe and 25 critical) as well as 25 healthy volunteer subjects participated in this study (Table 2(Tab. 2)). Of the 25 subjects in each group, 13 were male and 12 were female. The mean ages of the mild, severe, and critical patients and healthy volunteer group were 52.84±2.70, 51.08±2.40, 54.28±2.44, and 49.12±2.75, respectively (Table 2(Tab. 2)).

Expression of ZBP1, AIM2 and MDA5 genes in COVID-19 patients and healthy subjects

Our results showed a significant increase in the expression level of ZBP1 (P=0.0001), AIM2 (P<0.001), and MDA5 (P=0.003) genes in COVID-19 patients compared to the healthy control group (Figure 1(Fig. 1)).

Expression of ZBP1, AIM2 and MDA5 genes in COVID-19 patients based on the outcome of the disease

We found a significant increase in the expression level of ZBP1 gene in the critical (P<0.001) and severe (P=0.008) COVID-19 patients compared to the mild group (Figure 2A(Fig. 2)). We also found that AIM2 transcript expression level was significantly increased in the critical (P<0.001) and severe (P=0.0002) COVID-19 patients compared to the mild group (Figure 2B(Fig. 2)). Moreover, data analysis showed that the expression level of MDA5 gene was significantly higher in the critical (P=0.0006,) and severe (P= 0.003) COVID-19 patients compared to the mild group (Figure 2C(Fig. 2)).

Comparison of the expression levels of ZBP1, AIM2, and MDA5 genes between the males and females in different stages of COVID-19 patients

Remarkably, the mRNA levels of AIM2 were significantly elevated in the male critical (P=0.005) and severe (P=0.04) COVID-19 patients compared to female COVID-19 patients in the same stage (Figure 3B(Fig. 3)). Our results also showed a significant increase in the expression level of MDA5 gene in the male critical (P=0.0004) and severe (P=0.003) COVID-19 patients in comparison of the female critical and severe COVID-19 patients (Figure 3C(Fig. 3)). As the disease progressed, male ZBP1 mRNA expression was more than that of females; however, there was no significant difference in any stage of the disease (Figure 3A(Fig. 3)).

The expression levels of ZBP1, AIM2, and MDA5 genes in the male and female COVID-19 patient groups as well as healthy control group

The expression level of ZBP1 mRNA was significantly increased in the male critical patients compared to the mild ones (P=0.001) and healthy control subjects (P<0.001). Moreover, in severe COVID-19 patients, ZBP1 mRNA was significantly increased compared to the healthy control group (P=0.008) (Figure 4A(Fig. 4)). In addition, there were significant elevations in the levels of ZBP1 expression in the female critical (P=0.0001) and severe (P=0.003) COVID-19 patients compared to the healthy control group (Figure 4B(Fig. 4)).

We also found a significant increase in the expression level of AIM2 transcript in the male critical and severe COVID-19 patients compared to the mild (P<0.001 and P=0.04 respectively) and healthy control group (P<0.001 and P=0.02, respectively) (Figure 4C(Fig. 4)). There was also a significant increase in AIM2 mRNA in female patients with critical and mild levels (P=0.04). Additionally, we found a significant increase in the level of AIM2 transcript in the female critical (P<0.001) and severe (P=0.005) COVID-19 patients compared to the healthy control group (Figure 4D(Fig. 4)).

In addition, MDA5 transcript in male critical patients showed a significant increase compared to the severe (P=0.04), mild (P=0.01) and control groups (P= 0.001) (Figure 4E(Fig. 4)). In addition, a significant increase was seen in the level of MDA5 transcripts in the female critical and severe COVID-19 patients compared to the mild (P=0.001 and P=0.003, respectively) and healthy control group (P<0.001 and 0.001, respectively) (Figure 4F(Fig. 4)).

Discussion

SARS-CoV-2 is associated with COVID-19 global pandemic and has raised international concerns. It causes not only severe pneumonia and acute respiratory distress syndrome (ARDS) in vulnerable people, but also damage to many organs including the heart, kidney, pancreas, etc., and in severe cases, the disease can lead to death (Anka et al., 2021[1]; Mokhtari et al., 2020[28]). An antiviral defense initiates with the recognition of viruses by a wide range of PRRs (Kasuga et al., 2021[19]). SARS-CoV-2 could induce transcription of the genes encoding type I and III interferon (IFNs), as well as pro-inflammatory cytokines (Sampaio et al., 2021[36]). Despite the ability of IFNs to help clear the virus infection, aberrant production and impaired inflammasome activation, due to overexpression and overactivation, play a significant role in the pathogenesis of COVID-19 (Sharif-Zak et al., 2022[37]). Here, we determined and compared the expression levels of ZBP1, AIM2, and MDA5 genes transcript in different stages of COVID-19 disease as well as healthy control subjects.

The results revealed that the expression level of ZBP1 gene in COVID-19 patients significantly increased compared to the control group. Our results also revealed an ascending increase in the expression of ZBP1 mRNA from mild to critical stage in both male and female groups (Figure 2(Fig. 2)). ZBP1 is one of the important DNA sensors that can recognize DNA and RNA molecule of viruses when they are replicating or transcribing (Maelfait et al., 2017[25]), and it is bone fide inflammation mediator by its adaptor protein (Peng et al., 2021[32]). A study reported that ZBP1 signaling pathway was involved in necroptosis by TNF (Zhu et al., 2018[53]). Li et al. demonstrated that this signaling complex could be involved in the lung damage and cause exacerbation in the conditions of COVID-19 patients (Li et al., 2020[22]). Moreover, Peng et al. reported that ZPB1, as a primary response, stimulated the inflammatory signaling pathway that depends on ubiquitination and RIPK3's scaffolding ability. They also demonstrated that ZBP1 affected the production of inflammatory cytokines, such as IP-10 and CXCL10 in critical patients as a part of the cytokine storm (Peng et al., 2021[32]). Furthermore, it has been reported that ZBP1 is a vital gene expressed following infection of human and mice lung cell with an isolate of Middle East Respiratory Syndrome (MERS)-CoV and SARS-CoV-1 (Mamoor, 2020[26]). Kuriakose and colleagues also showed that ZBP1-mediated sensing PB1 and NP proteins of IAV triggered the inflammatory responses and cell death through RIPK1-RIPK3-caspase-8 (Kuriakose et al., 2016[21]). During infection with IAV in mice lacking ZBP1, inflammation and epithelial damages were reduced, which led to decreased mortality (Wang et al., 2008[44]). These findings indicate a positive association between the expression level of ZBP1 mRNA and disease severity and confirm our results, showing that high levels of ZBP expression could lead to the production of inflammatory cytokines, reduction of the immune system, and progression of the disease.

Our results also showed that the level of AIM2 transcript was significantly different between the COVID-19 patients and control group (Figure 4(Fig. 4)). In this regard, the results of Junqueira's study suggested that increasing the expression level of the AIM2 gene in COVID-19 patients, as one of the important molecules, might contribute to exacerbation of the condition of hospitalized patients (Junqueira et al., 2021[15]). Other studies indicated that AIM2 by processing Gradermin D protein, as one of the essential components of the inflammasome complex and apoptosis-associated Speck-like protein containing a caspase recruitment domain (ASC) (Pontelli et al., 2020[33]), activated pro-caspase 1, Interleukin-1β (IL1β) and IL18, and induced inflammation and programmed cell death (Cridland et al., 2012[6]). In another study, researchers showed that AIM2 might play a vital role in the immunopathogenesis of COVID-19 patients including primary pneumonia to respiratory failure and systemic disease. Moreover, they found a strong association between Gradermin D and inflammation caused by inflammasome in the severe COVID-19 patients compared to those with milder disease (Junqueira et al., 2021[15]). Accordingly, these results indicated that AIM2 signaling might contribute to an unbalanced immune response in COVID-19 patients characterized by enhanced inflammatory responses, reduced antiviral signaling, potentially leading to tissue damage or disease severity.

Here, we showed that the expression of the MDA5 gene in COVID-19 patients increased compared to the control group (Figure 1C(Fig. 1)). MDA5 expression plays an substantial role in inducing antiviral responses (Junior et al., 2019[13]). Studies revealed that interferons induced by PRR play an important role in inhibiting SARS-CoV-2 infection (Paludan and Mogensen, 2022[30]). Interestingly, MDA5 also plays a critical role in the two other severe coronavirus-infected diseases that happened in this century including SARS-1 and MERS (Xu, 2020[46]). In a study conducted by Sampaio and colleagues, it was found that the IFN type I and III response to SARS-CoV-2 depended on the MDA5, a sensor that is part of the detection process of SARS-CoV2 RNA (Sampaio et al., 2021[36]). Another research group demonstrated that MDA5 was the main sensor of SARS-CoV-2 in the lung cancer cell line, Calu-3 cells (Rebendenne et al., 2021[34]). It was noted that Wang et al. reported persistent overactivation of the MDA5 signaling in severe COVID-19 patients (Bader et al., 2022[3]), which confirms our findings. A study by Wang et al. concluded that the MDA5 signaling might be persistently over-activated in severe COVID-19 patients (Wang et al., 2021[41]), which confirming our data. Accordingly, this suggests that MDA5 expression might play a protective role in COVID-19 patients by causing interferon induction in the case of SARS-CoV-2 infection.

The increased expression of ZBP1, AIM2 and MDA5 genes can be interpreted in two ways. In the first case, the expression of these genes may increase as a result of corona virus infection, and in the second case, the person may become more susceptible to COVID-19 infection as a result of the increased expression of these genes.

The expression of ZBP1, AIM2, and MDA5 genes may increase as a result of corona virus infection. Influenza A virus (IAV)-induced expression of ZBP1 has been shown to be mediated by type-I IFN in BMDMs in vitro (Kuriakose et al., 2016[21]). Another group of researchers discovered that ZBP1 was significantly induced by pro-inflammatory mediators in lung tissues infected by IAV (Wang et al., 2019[43]). It has also been reported that IAV-induced type I IFN signaling is required for the ZBP1 upregulation (Kesavardhana et al., 2017[20]; Zheng and Kanneganti, 2020[51]). SARS-CoV-2 also infects blood monocytes, causing AIM2 inflammasomes to be activated (Junqueira et al., 2021[15]). During severe COVID-19, mitochondrial (mt) DNA and/or cell free (cf) DNA play an important role in triggering inflammatory responses and following cytokine storms, which can increase ISG mRNA such as AIM2. One study showed that oxidized mtDNA activates AIM2, which is unexpectedly activated by SARS-CoV-2 (Ferreira et al., 2021[8]). This dysregulation of mitochondrial function results in the formation of reactive oxygen species (ROS) and release of mtDNA into the cytosol of SARS-CoV-2- infected cells. ROS formation causes oxidization of mtDNA, and this oxidized mtDNA is a potent activator for AIM2 (Moriyama et al., 2020[29]). This increase in cfDNA in COVID-19 patients has been linked to the activation of the AIM2 inflammasome (Kaivola et al., 2021[16]). Furthermore, in the case of MDA5, a study reported that coronaviruses induce expression of MDA5, as previously demonstrated for mouse hepatitis virus (MHV) (Roth-Cross et al., 2008[35]). In addition, higher expression of MDA5 is triggered by SARS-CoV-2 infection, as reported by Loske et al. (2022[24]).

On the other hand, genetic variation in the regulatory and coding regions of host genes, including those involved in immune system response might play an important role in the pathogenesis and outcome of viral infections. In this regard, a gene cluster on chromosome 3 containing CCR1, CCR3, CXCR6, and CCR9, has been identified as a major risk factor for COVID-19 at the genome-wide level (Zeberg and Pääbo, 2020[48]). Furthermore, whole-exome sequencing revealed that loss-of-function variants of the X-chromosomal TLR7 in COVID-19 male patients could explain the predominance of COVID-19 (van der Made et al., 2020[40]). Variants at the TLR3 and IRF7 genes have also been linked to life-threatening COVID‐19 pneumonia and asymptomatic infection. Moreover, Zhang et al. found that at least 3.5 % of patients with life-threatening COVID-19 pneumonia had known or new genetic defects at eight of the candidate loci involved in the TLR3- and IRF7-dependent induction and amplification of type I IFNs (Zhang et al., 2020[49]).

Regarding the subjects' gender, data analysis showed that the expression level of AIM2 and MDA5 mRNA was significantly increased in the male critical and severe COVID-19 patients than females in the same stage. Moreover, the expression level of ZBP1, AIM2, and MDA5 genes was different between the male critical patients and male mild and healthy subjects (Figure 4A(Fig. 4)). In addition, the expression level of AIM2 and MDA5 genes was higher in the female severe and critical groups than healthy control groups (Figure 4D, F(Fig. 4)). A noticeable feature of the SARS-CoV-2 infection was the difference in the severity and outcome of COVID-19 disease between males and females (Galasso et al., 2020[9]; Jin et al., 2020[12]; Peckham et al., 2020[31]). Also, male patients tended to display more inflammation responses that contributes to the disease progression and outcome (Jin et al., 2020[12]; Peckham et al., 2020[31]). Furthermore, studies revealed that the odds of hospitalization, ICU admission, and mortality were nearly three times higher in male patients compared to females (Chan et al., 2020[4]). Although the underlying mechanisms are unclear, several factors have been speculated to account for the disparity including differences in biology, behavior, occupation, etc. (Galasso et al., 2020[9]). In addition, previous studies have shown that immune responses in females have been more effective than males against pathogens such as SARS-CoV-2 (Pontelli et al., 2020[33]; Singer, 2019[38]). This might be due to the effects of sex hormones, genes escaping X chromosome inactivation, skewed X chromosome inactivation, miRNAs encoded on the X chromosome, and the varied expression of DNA Methyl Transferase (DNMTs) in males and females (Migliore et al., 2021[27]). Another possible reason for the decreased transcription of MDA5 in the male subjects might be the role of SARS-CoV-2 proteins which could regulate antiviral responses. In this regard, researchers explain that the SARS-CoV-2 membrane protein (M) interacts with MDA5 and inhibits the production of type I and III IFNs by preventing the multiprotein complex containing MAVS, TRAF3, and TBK1 (Zheng et al., 2020[51]). In conclusion, the results of our study showed a steady increase in the expression of ZBP1, AIM2, and MDA5 genes transcript, indicating that ZBP1, AIM2, and MDA5 might play important roles in the severity and outcome of COVID-19 disease. Moreover, the expression level of ZBP1, AIM2, and MDA5 was higher in male patients than females, suggesting that the severity of COVID-19 disease in male patients compared with female ones might be related to the higher expression of AIM2 and MDA5. We recommend that further studies be conducted to clarify this issue.

Notes

Ramin Yaghobi and Jamal Sarvari (Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran; Phone: +9871-42652781, FAX: +9871-42710780, Email: sarvarij@sums.ac.ir) contributed equally as corresponding author.

DeclarationConflict of interest

All authors declare no conflict of interest.

Authors' contribution

Study concept: Sarvari J, Yaghobi R, Arab Zadeh SAM and Farokhnia M; Study design: Sarvari J, Yaghobi R and Ramezani A; Bench work: Arefinia N; Patients selection: Farokhnia M; Data analysis: Arefinia N and Ramezani A; Manuscript drafting: Arefinia N; Critical revision of the manuscript: Sarvari J, Yaghobi R, Arab Zadeh SAM, Farokhnia M and Ramezani A. All authors read and approved the final manuscript.

Ethical approval

Written informed consent was obtained from each participant, and the study was approved by the local Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.REC.1400.737).

Anka AU Tahir MI Abubakar SD Alsabbagh M Zian Z Hamedifar H Coronavirus disease 2019 (COVID‐19): an overview of the immunopathology, serological diagnosis and management Scand J Immunol 2021 93 4 e12998 Ansariniya H Seifati SM Zaker E Zare F Comparison of immune response between SARS, MERS, and COVID-19 infection, perspective on vaccine design and development Biomed Res Int 2021 2021 8870425 Bader SM Cooney JP Pellegrini M Doerflinger M Programmed cell death: the pathways to severe COVID-19? Biochem J 2022 479 609 628 Chan JF-W Yuan S Kok K-H To KK-W Chu H Yang J A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster Lancet 2020 395 10223 514 523 Chaplin DD Overview of the immune response J Allergy Clin Immunol 2010 125 2 S3 S23 Cridland JA Curley EZ Wykes MN Schroder K Sweet MJ Roberts TL The mammalian PYHIN gene family: phylogeny, evolution and expression BMC Evol Biol 2012 12 140 Daniels BP Kofman SB Smith JR Norris GT Snyder AG Kolb JP The nucleotide sensor ZBP1 and kinase RIPK3 induce the enzyme IRG1 to promote an antiviral metabolic state in neurons Immunity 2019 50 1 64 76 Ferreira AC Soares VC de Azevedo-Quintanilha IG Dias SdSG Fintelman-Rodrigues N Sacramento CQ SARS-CoV-2 engages inflammasome and pyroptosis in human primary monocytes Cell Death Discov 2021 7 1 43 Galasso V Pons V Profeta P Becher M Brouard S Foucault M Gender differences in COVID-19 attitudes and behavior: Panel evidence from eight countries Proc Natl Acad Sci U S A 2020 117 27285 27291 Ghoreshi Z-a Nakhaee M Samie M Zak MS Arefinia N Innate immune sensors for detecting nucleic acids during infection LaboratoriumsMedizin 2022 46 155–64 Guan W-j Ni Z-y Hu Y Liang W-h Ou C-q He J-x Clinical characteristics of coronavirus disease 2019 in China N Engl J Med 2020 382 1708 1720 Jin J-M Bai P He W Wu F Liu X-F Han D-M Gender differences in patients with COVID-19: focus on severity and mortality Front Public Health 2020 8 152 Junior AGD Sampaio NG Rehwinkel J A balancing act: MDA5 in antiviral immunity and autoinflammation Trends Microbiol 2019 27 75 85 Junqueira C Crespo  Ranjbar S de Lacerda LB Lewandrowski M Ingber J FcγR-mediated SARS-CoV-2 infection of monocytes activates inflammation Nature 2022 606 7914 576 584 Junqueira C Crespo  Ranjbar S Lewandrowski M Ingber J de Lacerda LB SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release medRxiv [Preprint] 8 Mar 2021 2021.03.06.21252796 10.1101/2021.03.06.21252796 Kaivola J Nyman TA Matikainen S Inflammasomes and SARS-CoV-2 infection Viruses 2021 13 12 2513 Kang D-c Gopalkrishnan RV Lin L Randolph A Valerie K Pestka S Expression analysis and genomic characterization of human melanoma differentiation associated gene-5, mda-5: a novel type I interferon-responsive apoptosis-inducing gene Oncogene 2004 23 1789 1800 Kang D-c Gopalkrishnan RV Wu Q Jankowsky E Pyle AM Fisher PB mda-5: An interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties Proc Natl Acad Sci U S A 2002 99 637 642 Kasuga Y Zhu B Jang K-J Yoo J-S Innate immune sensing of coronavirus and viral evasion strategies Exp Mol Med 2021 53 723 736 Kesavardhana S Kuriakose T Guy CS Samir P Malireddi RS Mishra A ZBP1/DAI ubiquitination and sensing of influenza vRNPs activate programmed cell death J Exp Med 2017 214 2217 2229 Kuriakose T Man SM Subbarao Malireddi R Karki R Kesavardhana S Place DE ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways Sci Immunol 2016 1 2 aag2045 aag2aag Li S Zhang Y Guan Z Li H Ye M Chen X SARS-CoV-2 triggers inflammatory responses and cell death through caspase-8 activation Signal Transduct Target Ther 2020 5 1 235 Liu T Feng M Wen Z He Y Lin W Zhang M Comparison of the characteristics of cytokine storm and immune response induced by SARS-CoV, MERS-CoV, and SARS-CoV-2 infections J Inflamm Res 2021 14 5475 5487 Loske J Röhmel J Lukassen S Stricker S Magalhães VG Liebig J Pre-activated antiviral innate immunity in the upper airways controls early SARS-CoV-2 infection in children Nat Biotechnol 2022 40 319 324 Maelfait J Liverpool L Bridgeman A Ragan KB Upton JW Rehwinkel J Sensing of viral and endogenous RNA by ZBP 1/DAI induces necroptosis EMBO J 2017 36 2529 2543 Mamoor S Transcriptional induction of nucleic acid sensors during coronavirus infection OSF preprints 2020 10.31219/osf.io/b8cfk Migliore L Nicolì V Stoccoro A Gender specific differences in disease susceptibility: the role of epigenetics Biomedicines 2021 9 6 652 Mokhtari T Hassani F Ghaffari N Ebrahimi B Yarahmadi A Hassanzadeh G COVID-19 and multiorgan failure: A narrative review on potential mechanisms J Mol Histol 2020 51 613 628 Moriyama M Nagai M Maruzuru Y Koshiba T Kawaguchi Y Ichinohe T Influenza virus-induced oxidized DNA activates inflammasomes Iscience 2020 23 7 101270 Paludan SR Mogensen TH Innate immunological pathways in COVID-19 pathogenesis Sci Immunol 2022 7 67 eabm5505 Peckham H de Gruijter NM Raine C Radziszewska A Ciurtin C Wedderburn LR Male sex identified by global COVID-19 meta-analysis as a risk factor for death and ITU admission Nat Commun 2020 11 1 6317 Peng R Wang-Kan X Idorn M Zhou FY Orozco SL McCarthy J ZBP1 induces inflammatory signaling via RIPK3 and promotes SARS-CoV-2-induced cytokine expression bioRxiv [Preprint] Oct 2012 10.1101/2021.10.01.462460 Pontelli MC Castro IA Martins RB Veras FP La Serra L Nascimento DC Infection of human lymphomononuclear cells by SARS-CoV-2 bioRxiv [Preprint] 7 Aug 2020 7:2020.07.28.225912 10.1101/2020.07.28.225912 Rebendenne A Chaves Valadão AL Tauziet M Maarifi G Bonaventure B McKellar J SARS-CoV-2 triggers an MDA-5-dependent interferon response which is unable to control replication in lung epithelial cells J Virol 2021 95 8 e02415 e02420 Roth-Cross JK Bender SJ Weiss SR Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/microglia J Virol 2008 82 20 9829 9838 Sampaio NG Chauveau L Hertzog J Bridgeman A Fowler G Moonen JP The RNA sensor MDA5 detects SARS-CoV-2 infection Sci Rep 2021 11 1 13638 Sharif-Zak M Abbasi-Jorjandi M Asadikaram G Rezazadeh-Jabalbarzi M Rashidinejad H Khajepour F CCR2 and DPP9 expression in the peripheral blood of COVID-19 patients: Influences of the disease severity and gender Immunobiology 2022 227 2 152184 Singer BD A practical guide to the measurement and analysis of DNA methylation Am J Respir Cell Mol Biol 2019 61 417 428 Thapa RJ Ingram JP Ragan KB Nogusa S Boyd DF Benitez AA DAI senses influenza A virus genomic RNA and activates RIPK3-dependent cell death Cell Host Microbe 2016 20 674 681 van der Made CI Simons A Schuurs-Hoeijmakers J van den Heuvel G Mantere T Kersten S Presence of genetic variants among young men with severe COVID-19 JAMA 2020 324 663 673 Wang G Wang Q Wang Y Liu C Wang L Chen H Presence of anti-MDA5 antibody and its value for the clinical assessment in patients with COVID-19: a retrospective cohort study Front Immunol 2021 12 791348 Wang L Sun L Byrd KM Ko C-C Zhao Z Fang J AIM2 inflammasome's first decade of discovery: focus on oral diseases Front Immunol 2020 11 1487 Wang Y Hao Q Florence JM Jung B-G Kurdowska AK Samten B Influenza virus infection induces ZBP1 expression and necroptosis in mouse lungs Front Cell Infect Microbiol 2019 9 286 Wang Z Choi MK Ban T Yanai H Negishi H Lu Y Regulation of innate immune responses by DAI (DLM-1/ZBP1) and other DNA-sensing molecules Proc Natl Acad Sci U S A 2008 105 5477 5482 WHO, World Health Organization COVID-19 clinical management: living guidance, 25 January 2021 2021 Geneva WHO Xu Q MDA5 should be detected in severe COVID-19 patients Med Hypotheses 2020 143 109890 Zalinger ZB Elliott R Rose KM Weiss SR MDA5 is critical to host defense during infection with murine coronavirus J Virol 2015 89 12330 12340 Zeberg H Pääbo S The major genetic risk factor for severe COVID-19 is inherited from Neanderthals Nature 2020 587 610–2 Zhang Q Bastard P Liu Z Le Pen J Moncada-Velez M Chen J Inborn errors of type I IFN immunity in patients with life-threatening COVID-19 Science 2020 370 6515 eabd4570 Zhang T Yin C Boyd DF Quarato G Ingram JP Shubina M Influenza virus Z-RNAs induce ZBP1-mediated necroptosis Cell 2020 180 1115 1129 Zheng M Kanneganti TD The regulation of the ZBP1‐NLRP3 inflammasome and its implications in pyroptosis, apoptosis, and necroptosis (PANoptosis) Immunol Rev 2020 297 26 38 Zheng Y Zhuang MW Han L Zhang J Nan ML Zhan P Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) membrane (M) protein inhibits type I and III interferon production by targeting RIG-I/MDA-5 signaling Signal Transduct Target Ther 2020 5 1 299 Zhu K Liang W Ma Z Xu D Cao S Lu X Necroptosis promotes cell-autonomous activation of proinflammatory cytokine gene expression Cell Death Dis 2018 9 500 The sequences of primers used for RT-qPCR in the study Demographical characteristics of the three study groups and healthy volunteer control group A) Comparison of the expression of ZBP1 in the peripheral blood samples between total COVID-19 patients and the control group. B) Comparison of the expression of AIM2 in the peripheral blood samples between total COVID-19 patients and the control group. C) Comparison of the expression of MDA5 in the peripheral blood samples between total COVID-19 patients and the control group. Data are presented as fold change. ns: not significant. Significant difference in different groups (p < 0.05). Comparison of the expression of ZBP1 A), AIM2 B), and MDA5 C) genes in different stages of the COVID-19 patients. Data are presented as fold change. ns: not significant. There are difference between specified groups (p < 0.05). Comparison of ZBP1 A), AIM2 B), and MDA5 C) gene expression levels between male and female patients in different outcomes of the disease. Data are presented as fold change. ns: not significant. There are differences between specified groups (p < 0.05). The expression level of ZBP1, AIM2, and MDA5 genes in male and female patients with COVID-19 according to the outcome of disease and gender. A) The expression level of ZBP1 gene in male COVID-19 patients. B) The expression level of ZBP1 gene in female COVID-19 patients. C) The expression level of AIM2 gene in male COVID-19 patients. D) The expression level of AIM2 gene in female COVID-19 patients. E) The expression level of MDA5 gene in male COVID-19 patients. F) The expression level of MDA5 gene in female COVID-19 patients. Data are presented as fold change. ns: not significant. There is a difference between the specified groups (p < 0.05).